Novel formulations

ABSTRACT

According to the invention there is provided inter alia an aqueous liquid pharmaceutical formulation comprising insulin or an insulin analogue, ionic zinc, a chelating agent and polysorbate 80.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/720,017, filed Sep. 29, 2017, which is a continuation-in-part ofInternational Patent Application No. PCT/GB2017/051254 filed May 5,2017, which claims priority under 35 U.S.C. § 119 or 365 to UnitedKingdom Application No. 1607918.8, filed May 6, 2016, each of which ishereby incorporated by reference in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing in ASCIItext file (Name: 6662_0012 Sequence_Listing_.txt; Size: 1.67 KB; andDate of Creation: Sep. 28, 2017) filed with the application is hereinincorporated by reference in its entirety.

FIELD OF THE INVENTION

This invention relates inter alia to rapid acting aqueous liquidcompositions of insulin and insulin analogues. Such compositions aresuitable for the treatment of subjects suffering from diabetes mellitus,especially Type I diabetes mellitus.

BACKGROUND OF THE INVENTION

Diabetes mellitus (“diabetes”) is a metabolic disorder associated withpoor control of blood sugar levels leading to hypo or hyperglycemia.Untreated diabetes can lead to serious microvascular and macrovascularcomplications including coronary artery disease, peripheral arterydisease, stroke, diabetic nephropathy, neuropathy and retinopathy. Thetwo main types of diabetes are (i) Type 1 diabetes resulting from thepancreas not producing insulin for which the usual treatment is insulinreplacement therapy and (ii) Type 2 diabetes where patients eitherproduce insufficient insulin or have insulin resistance and for whichtreatments include insulin sensitising agents (such as metformin orpioglitazone), traditional insulin secretagogues (such assulfonylureas), SGLT2 inhibitors (such as dapagliflozin, canagliflozinand empagliflozin) which reduce glucose absorption in the kidneys and sopromote glucose excretion, GLP-1 agonists (such as exenatide anddulaglutide) which stimulate insulin release from pancreatic beta cellsand DPPIV inhibitors (such as sitagliptin or vildagliptin) which inhibitbreakdown of GLP-1 leading to increased insulin secretion. Patients withType 2 diabetes may eventually require insulin replacement therapy.

For patients requiring insulin replacement therapy, a range oftherapeutic options are possible. The use of recombinant human insulinhas in recent times been overtaken by use of insulin analogues whichhave modified properties, for example, are longer acting or fasteracting than normal insulin. Thus, a common regimen for a patientinvolves receiving a long acting basal insulin supplemented by a rapidacting insulin around mealtimes.

Insulin is a peptide hormone formed of two chains (A chain and B chain,respectively 21 and 30 amino acids in length) linked via disulfidebridges. Insulin normally exists at neutral pH in the form of a hexamer,each hexamer comprising three dimers bound together by zinc ions.Histidine residues on the insulin are known to be involved in theinteraction with the zinc ions. Insulin is stored in the body in thehexameric form but the monomer form is the active form. Traditionally,therapeutic compositions of insulin have also been formulated inhexameric form in the presence of zinc ions. Typically, there areapproximately three zinc cations per one insulin hexamer. It has beenappreciated that the hexameric form is absorbed from the injection siteconsiderably more slowly than the monomeric and dimeric form. Therefore,a faster onset of insulin action can be achieved if the hexameric formis destabilised allowing a more rapid dissociation of the zinc-boundhexamer into dimers and monomers in the subcutaneous space followinginjection. Three insulin analogues have been genetically engineered withthis principle in mind. A first is insulin lispro (Humalog®) in whichresidues 28 and 29 of the B chain (Pro and Lys respectively) arereversed, a second is insulin aspart (NovoLog®) in which residue 28 ofthe B chain, normally Pro, is replaced by Asp and a third is insulinglulisine (Apidra®) in which residue 3 of the B chain, normally Asn, isreplaced by Lys and residue 29 of the B chain, normally Lys, is replacedby Glu.

Whilst the existing rapid acting insulin analogues can achieve a morerapid onset of action, it has been appreciated that an even more rapidacting (“ultra rapid acting”) insulins can be achieved by removing thezinc cations from insulin altogether. Unfortunately, the consequence ofthe hexamer dissociation is typically a considerable impairment ininsulin stability both with respect to physical stability (e.g.stability to aggregation) and chemical stability (e.g. stability todeamidation). For example, monomeric insulin or insulin analogues havinga rapid onset of action are known to aggregate and become physicallyunstable very rapidly because the formulation of insoluble aggregatesproceeds via monomers of insulin. Various approaches to addressing thisproblem have been described in the art:

U.S. Pat. No. 5,866,538 (Norup) describes insulin preparations ofsuperior chemical stability comprising human insulin or an analogue orderivative thereof, glycerol and/or mannitol and 5 to 100 mM of ahalogenide (e.g. NaCl).

U.S. Pat. No. 7,205,276 (Boderke) addresses the stability problemsassociated with preparing zinc free formulations of insulin and insulinderivatives and analogues and describes an aqueous liquid formulationcomprising at least one insulin derivative, at least one surfactant,optionally at least one preservative and optionally at least one of anisotonicizing agent, a buffer and an excipient, wherein the formulationis stable and free from or contains less than 0.4% by weight of zincbased on the insulin content of the formulation. The preferredsurfactant appears to be polysorbate 20 (polyoxyethylene (20) sorbitanmonolaurate).

US2008/0194461 (Maggio) describes formulations of peptides andpolypeptides including insulin which contain an alkylglycoside, whichcomponent is said to reduce aggregation and immunogenicity.

WO2012/006283 (Pohl) describes formulations containing insulin togetherwith a zinc chelator such as ethylenediaminetetraacetate (EDTA).Modulating the type and quantity of EDTA is said to change the insulinabsorption profile. Calcium EDTA is the preferred form of EDTA since itis said to be associated with reduced pain at the injection site and isless likely to remove calcium from the body. Preferred formulations alsocontain citrate which is said to further enhance absorption and toimprove the chemical stability of the formulation.

US2010/0227795 (Steiner) describes a composition comprising insulin, adissociating agent such as citric acid or sodium citrate, and a zincchelator such as EDTA wherein the formulation has a physiological pH andis a clear aqueous solution. The formulations are said to have improvedstability and rapid onset of action.

WO2015/120457 (Wilson) describes stabilized ultra-rapid acting insulinformulations comprising insulin in combination with a zinc chelator suchas EDTA, a dissolution/stabilization agent such as citric acid, amagnesium salt, a zinc compound and optionally additional excipients.

Further approaches to accelerating the absorption and effect of insulinthrough the use of specific accelerating additives have been described:

WO_(91/09617) (Jorgensen) reports that nicotinamide or nicotinic acid ora salt thereof increases the speed of absorption of insulin from aqueouspreparations administered parenterally.

WO2010/149772 (Olsen) describes a formulation comprising insulin, anicotinic compound and arginine. The presence of arginine is said toimprove the chemical stability of the formulation.

WO2015/171484 (Christe) described rapid acting formulations of insulinwherein onset of action and/or absorption of insulin is faster due tothe presence of treprostinil.

US2013/0231281 (Soula) describes an aqueous solution compositioncomprising insulin or an insulin analogue and at least oneoligosaccharide whose average degree of polymerisation is between 3 and13 and whose polydispersity index is above 1.0, said oligosaccharidehaving partially substituted carboxyl functional groups, theunsubstituted carboxyl functional groups being salifiable. Such aformulation is said to be rapid acting.

It would be desirable if analogues or formulations of insulin wereavailable which were ultra rapid acting, thus, more closely matching theactivity of physiological insulin. There also remains a need in the artto provide further, and preferably improved, formulations of insulin andinsulin analogues which are rapid acting and stable.

SUMMARY OF THE INVENTION

According to the invention there is provided an aqueous liquidpharmaceutical formulation comprising insulin or an insulin analogue,ionic zinc, a chelating agent and polysorbate 80 (“the formulation ofthe invention”).

The formulation of the invention may be used in treatment of subjectssuffering from diabetes mellitus, particularly Type 1 diabetes mellitusespecially for administration at meal times.

As can be seen from the accompanying examples, formulations of theinvention are significantly more stable than corresponding formulationswithout polysorbate 80 or corresponding formulations in whichpolysorbate 80 is substituted by another non-ionic surfactant. Theformulations are expected to be more rapidly acting than correspondingformulations which do not contain a chelating agent.

Description of the Sequence Listing:

-   -   SEQ ID NO: 1: A chain of human insulin    -   SEQ ID NO: 2: B chain of human insulin    -   SEQ ID NO: 3: B chain of insulin lispro    -   SEQ ID NO: 4: B chain of insulin aspart    -   SEQ ID NO: 5: B chain of insulin glulisine

DETAILED DESCRIPTION OF THE INVENTION

As used herein, “insulin” refers to native human insulin having an Achain and a B chain as set out in SEQ ID NOs. 1 and 2 and containing andconnected by disulfide bridges as in the native molecule (Cys A6-CysA11, Cys B7 to Cys A7 and Cys-B19-Cys A20). Insulin is suitablyrecombinant insulin.

“Insulin analogue” refers to an analogue of insulin which is an insulinreceptor agonist and has a modified amino acid sequence, such ascontaining 1 or 2 amino acid changes in the sequence of the A or B chain(especially the B chain). Exemplary insulin analogues include fasteracting analogues such as insulin lispro, insulin aspart and insulinglulisine. These forms of insulin have the human insulin A chain butvariant B chains—see SEQ ID NOs. 3-5. Further faster acting analoguesare described in EP0214826, EP0375437 and EP0678522 the contents ofwhich are herein incorporated by reference in their entirety.

In one embodiment the insulin or insulin analogue is recombinant humaninsulin. In another embodiment it is insulin lispro. In anotherembodiment it is insulin aspart. In another embodiment it is insulinglulisine.

The term “aqueous pharmaceutical formulation”, as used herein, refers toa formulation suitable for therapeutic use in which the aqueouscomponent is or comprises water, preferably distilled water, deionizedwater, water for injection, sterile water for injection orbacteriostatic water for injection. The aqueous pharmaceuticalformulations of the invention are solution formulations in which allcomponents are dissolved in water.

The concentration of insulin or insulin analogue in the formulation willtypically be in the range 10-1000 U/ml, such as 50-500 U/ml e.g. 50-200U/ml. An exemplary formulation contains insulin or insulin analogue at aconcentration of 100 U/ml (around 3.6 mg/ml). Another range of interestis 500-1000 U/ml e.g. 800-1000 U/ml and another exemplary formulationcontains insulin or insulin analogue at a concentration of 1000 U/ml(around 36 mg/ml).

The formulations of the invention contain ionic zinc i.e. Zn²⁺ ions. Thesource of the ionic zinc will typically be a water soluble zinc saltsuch as ZnCl₂, ZnO, ZnSO₄, Zn(NO₃)₂ or Zn(acetate)₂ and most suitablyZnCl₂ or ZnO.

The concentration of the ionic zinc in the formulation will typically bemore than 0.05% e.g. more than 0.1% e.g. more than 0.2% e.g. more than0.25% by weight of zinc based on the weight of insulin or insulinanalogue in the formulation, for example 0.25-1%, e.g. 0.35-0.75%, e.g.0.45-0.6% by weight of zinc based on the weight of insulin or insulinanalogue in the formulation. For the purpose of the calculation theweight of counter ion to zinc is excluded. The concentration of theionic zinc in the formulation will (for example, for a formulationcontaining 100 U/ml of insulin or insulin analogue) typically be morethan 0.015 mM e.g. more than 0.03 mM e.g. more than 0.06 mM, more than0.09 mM or more than 0.12 mM. Thus, the concentration of the ionic zincin the formulation will typically be more than 0.15 mM, for example0.15-0.60 mM, e.g. 0.20-0.45 mM, e.g. 0.25-0.35 mM.

In a formulation e.g. containing 1000 U/ml of insulin or insulinanalogue the concentration of the ionic zinc will typically be more than0.15 mM e.g. more than 0.3 mM e.g. more than 0.6 mM, more than 0.9 mM ormore than 1.2 mM. Thus, the concentration of the ionic zinc in theformulation may be more than 1.5 mM, for example 1.5-6.0 mM, e.g.2.0-4.5 mM, e.g. 2.5-3.5 mM.

The formulations of the invention contain a chelating agent. Chelatingagents should be capable of complexing ionic zinc and typically willhave a logK metal binding stability constant with respect to zincbinding of at least 4.5 as determined at 25° C. Metal binding stabilityconstants listed in the National Institute of Standards and Technologyreference database 46 (Critically Selected Stability Constants of MetalComplexes) can be used. The database typically lists logK constantsdetermined at 25° C. Therefore, the suitability of a chelating agent forthe present invention can be determined based on its logK metal bindingstability constant with respect to zinc binding, as measured at 25° C.and as quoted by the database. Exemplary chelating agents includepolydendate organic anions. A preferred chelating agent is EDTA(logK=14.5). Further examples include citrate (logK=4.93), EGTA(logK=12.6), pyrophosphate (logK=8.71) and alginate (logK=6.91). Afurther preferred chelating agent is citrate. Other possible chelatingagents include substances that can contribute a lone pair of electronsor electron density for interaction with ionic zinc such as polydentateamines including ethylenediamine and aromatic or heteroaromaticsubstances especially those comprising an imidazole moiety such ashistidine (log K =6.51).

The most suitable concentration of the chelating agent will depend onthe agent and its logK value and will typically be in the range 0.1-100mM e.g. 0.1-50 mM.

For example, the concentration of the chelating agent in the formulationmay typically be in the range 0.1-2 mM, e.g. 0.2-1 mM, e.g. 0.3-1 mM,more preferably 0.3-0.5 mM, more preferably around 0.4 mM, especiallywhen the chelating agent is EDTA. For example, the concentration of thechelating agent in the formulation in the formulation may typically bein the range 2.5-50 mM, e.g. 5-50 mM, more preferably 5-30 mM, morepreferably around 20 mM (e.g. 22 mM), especially when the chelatingagent is citrate or histidine and especially for insulin or insulinanalogue 100 U/ml formulations. Suitably the concentration of the zincbinding species in the formulation is 10-50 mM e.g.30-50 mM e.g. 40-50mM, more preferably around 44 mM when the zinc binding species iscitrate or histidine for insulin or insulin analogue 1000 U/mlformulations.

Chelating agents such as EDTA, citrate, EGTA, pyrophosphate or alginatemay be employed as the free acid or a salt form, such as a salt formwith sodium or calcium ions, especially sodium ions.

Chelating agents which have acid forms (e.g. citric acid) may beintroduced into the aqueous formulations of the invention in the form ofa salt of the acid, such as a sodium salt (e.g. sodium citrate).Alternatively, they can be introduced in the form of the acid withsubsequent adjustment of pH to the required level.

A mixture of chelating agents may be employed, although a singlechelating agent is preferred.

Suitably the molar ratio of ionic zinc to chelating agent in theformulation is in the range 1.0:0.8 to 1:500 e.g.1.0:0.8 to 1:100.

For example, a suitable range of ionic zinc to chelating agent is1.0:0.8 to 1.0:2.0, e.g. 1.0:0.95 to 1.0:1.5, e.g. 1.0:1.0 to 1.0:1.4,especially for EDTA as chelating agent. The suitable molar ratio can beadapted accordingly for chelating agents with lower logK dissociationconstants than EDTA with respect to Zn binding. For example, anothersuitable molar ratio of ionic zinc to chelating agent is 1:20-1:100,e.g. 1:40-1:90, e.g. 1:60-1:80, especially for citrate or histidine aschelating agent.

For example, a formulation containing 100 U/ml of insulin or insulinanalogue may contain around 0.3 mM of ionic zinc (i.e. around 19.5 pg/m1of ionic zinc, i.e. around 0.54% by weight of zinc based on the weightof insulin or insulin analogue in the formulation) and around 0.3-0.4 mMchelating agent (especially EDTA).

For example, a formulation containing 100 U/ml of insulin or insulinanalogue may contain around 0.3 mM of ionic zinc (i.e. around 19.5 μg/mlof ionic zinc, i.e. around 0.54% by weight of zinc based on the weightof insulin or insulin analogue in the formulation) and around 20 mM(e.g. 22 mM) chelating agent (especially citrate).

The formulations of the invention contain polysorbate 80 (also known aspolyoxyethylene (20) sorbitan monooleate) which is a non-ionicsurfactant. Thus, polysorbate 80 is a mono ester formed from oleic acidand polyoxyethylene (20) sorbitan in which the number 20 indicates thenumber of oxyethylene groups in the molecule. Polysorbate 80 is knownunder a range of brand names including in particular Tween 80, and alsoAlkest TW 80.

The concentration of the polysorbate 80 in the formulation willtypically be in the range 1-800 μg/ml, e.g. 1-500 μp/ml, e.g. 5-200μg/ml, such as 5-50 μp/m1 especially around 20 μg/ml. For higherconcentrations of insulin compound e.g. 500-1000 U/ml a higherconcentration of polysorbate 80 may be suitable e.g. 300-600 μg/ml, suchas 400-500 μg/ml especially around 500 μp/ml.

Suitably the pH of the aqueous formulations of the invention is in therange 5.5-9.0 especially 6.5-8.0 e.g. 7.0-7.5. In order to minimiseinjection pain the pH is preferably close to physiological pH (around pH7.4). Another pH range of interest is 7.6-8.0 e.g. around 7.8.

Suitably, the composition of the invention comprises a buffer in orderto stabilise the pH of the formulation, which can also be selected toenhance protein stability. In one embodiment, a buffer is selected tohave a pKa close to the pH of the composition; for example histidine issuitably employed as a buffer when the pH of the composition is in therange 5.0-7.0. Such a buffer may be employed in a concentration of 0.5-5mM e.g. 1-2 mM. If histidine is included in the formulation as achelating agent it will also have a buffering role at this pH. Ifcitrate is included in the formulation as a chelating agent it may alsohave a buffering role. As another example, phosphate is suitablyemployed as a buffer when the pH of the composition is in the range6.1-8.1. Such a buffer may be employed in a concentration of 0.5-3 mMe.g. 0.5-2 mM. Alternatively, in another embodiment, the formulation ofthe invention is further stabilised as disclosed in WO2008/084237(herein incorporated by reference in its entirety), which describes aformulation comprising a protein and one or more additives,characterised in that the system is substantially free of a conventionalbuffer, i.e. a compound with an ionisable group having a pKa within 1unit of the pH of the formulation at the intended temperature range ofstorage of the composition, such as 25° C. In this embodiment, the pH ofthe formulation is set to a value at which the formulation has maximummeasurable stability with respect to pH; the one or more additives(displaced buffers) are capable of exchanging protons with the insulinor insulin analogue and have pKa values at least 1 unit more or lessthan the pH of the formulation at the intended temperature range ofstorage of the formulation. The additives may have ionisable groupshaving pKa between 1 to 5 pH units, preferably between 1 to 3 pH units,most preferably from 1.5 to 2.5 pH units, of the pH of the aqueousformulation at the intended temperature range of storage of thecomposition (e.g. 25° C.). Such additives may typically be employed at aconcentration of 0.5-10 mM e.g. 2-5 mM.

The ionic strength of a formulation may be calculated according to theformula

${I:I} = {0.5 \times {\sum\limits_{X = 1}^{n}{c_{x}z_{x}^{2}}}}$

in which c_(x) is molar concentration of ion x (mol L⁻¹), z_(x) is theabsolute value of the charge of ion x and the sum covers all ions (n)present in the composition. The contribution of the insulin or insulinanalogue itself should be ignored for the purposes of the calculation.For zwitterions the absolute value of the charge is the total chargeexcluding polarity, e.g. for glycine the possible ions have absolutecharge of 0, 1 or 2 and for aspartate the possible ions have absolutecharge of 0, 1, 2 or 3. The ionic strength of the formulation issuitably kept to a minimum level since higher ionic strengthformulations are less stable than lower ionic strength formulations.

In general, the ionic strength of the formulation is suitably in therange of around 1 mM up to around 500 mM.

Suitably the ionic strength is less than 40 mM, e.g. less than 20 mM,e.g. less than 10 mM.

When the insulin analogue is insulin lispro, the ionic strength of theformulation is suitably kept to a minimum level since higher ionicstrength formulations are less stable than lower ionic strengthformulations. Suitably the ionic strength taking account of ions in theformulation except for the zinc binding species and the insulin analogueis less than 40 mM, e.g. less than 20 mM, e.g. less than 10 mM such as1-10 mM.

When the insulin analogue is insulin aspart at a concentration of >500U/ml (e.g. 1000 U/ml), the ionic strength of the formulation is suitablykept to a minimum level since higher ionic strength formulations areless stable than lower ionic strength formulations. Suitably the ionicstrength taking account of ions in the formulation except for the zincbinding species and the insulin analogue is less than 40 mM, e.g. lessthan 20 mM, e.g. less than 10 mM.

When the insulin analogue is insulin aspart at a concentration of 500U/ml or less (e.g. 100 U/ml), the ionic strength of the formulation maybe high. Suitably the ionic strength taking account of ions in theformulation except for the zinc binding species and the insulin analogueis more than 50 mM, e.g. more than 100 mM, e.g. 50-500 mM or 100-500 mMor 100-300 mM such as around 150 mM.

The aqueous formulations of the present invention cover a wide range ofosmolarity, including hypotonic, isotonic and hypertonic compositions.Preferably, the formulations of the invention are substantiallyisotonic. Suitably the osmolarity of the formulation is selected tominimize pain according to the route of administration e.g. uponinjection. Preferred formulations have an osmolarity in the range ofabout 200 to about 500 mOsm/L. Preferably, the osmolarity is in therange of about 250 to about 350 mOsm/L. More preferably, the osmolarityis about 300 mOsm/L.

Tonicity of the formulation may be adjusted with a tonicity modifyingagent. Tonicity modifying agents may be charged or uncharged anduncharged tonicity modifying agents are preferred. Examples of chargedtonicity modifying agents include salts such as a combination of sodium,potassium, magnesium or calcium ions, with chloride, sulfate, carbonate,sulfite, nitrate, lactate, succinate, acetate or maleate ions(especially sodium chloride or sodium sulphate, particularly sodiumchloride). Amino acids such as arginine, glycine or histidine may alsobe used for this purpose. Examples of uncharged tonicity modifyingagents include sugars, sugar alcohols and other polyols, such astrehalose, sucrose, mannitol, glycerol, 1,2-propanediol, raffinose,lactose, dextrose, sorbitol or lactitol (especially trehalose, mannitol,glycerol or 1,2-propanediol, particularly glycerol).

The formulations of the invention can optionally include preservative,preferably phenol, m-cresol, chlorocresol, benzyl alcohol,propylparaben, methylparaben, benzalkonium chloride or benzethoniumchloride.

The formulations of the invention may optionally comprise nicotinamide.The presence of nicotinamide may further increase the speed of onset ofaction of insulin formulated in compositions of the invention. Suitably,the concentration of nicotinamide is in the range 10-150 mM, preferablyin the range 20-100 mM, such as around 80 mM.

The formulations of the invention may optionally comprise nicotinic acidor a salt thereof. The presence of nicotinic acid or a salt thereof mayalso further increase the speed of onset of action of insulin formulatedin compositions of the invention. Suitably, the concentration ofnicotinic acid or a salt thereof is in the range 10-150 mM, preferablyin the range 20-100 mM, such as around 80 mM. Example salts includemetal salts such as sodium, potassium and magnesium salts.

Typically one of nicotinamide and nicotinic acid (or as salt thereof)may be included in the formulation but not both.

Formulations of the invention may optionally include other beneficialcomponents including stabilising agents. For example amino acids such asarginine and proline may be included which may have stabilisingproperties.

In an embodiment of the invention the formulations are free of acidsselected from glutamic acid, ascorbic acid, succinic acid, asparticacid, maleic acid, fumaric acid, adipic acid and acetic acid and arealso free from the corresponding ionic forms of these acids. In anembodiment of the invention the formulations are free of citric acid andare also free from the corresponding ionic forms of this acid.

In an embodiment of the invention the formulations are free of protamineand protamine salts.

In an embodiment of the invention the formulations are free of magnesiumions.

In an embodiment of the invention the formulations are free of calciumions.

Suitably the formulations of the invention are sufficiently stable thatthe concentration of high molecular weight species remains low uponextended storage.

The term “high molecular weight species” as used herein, refers to anyirreversibly formed component of the protein content which has anapparent molecular weight at least about double the molecular weight ofthe parent insulin or insulin analogue, as detected by a suitableanalytical method, such as size-exclusion chromatography. That is, highmolecular weight species are multimeric aggregates of the parent insulinor insulin analogue. The multimeric aggregates may comprise the parentprotein molecules with considerably altered conformation or they may bean assembly of the parent protein units in the native or near-nativeconformation. The determination of high molecular weight species can bedone using methods known in the art, including size exclusionchromatography, electrophoresis, analytical ultracentrifugation, lightscattering, dynamic light scattering, static light scattering and fieldflow fractionation.

Suitably the formulations of the invention are sufficiently stable thatthey remain substantially free of visible particles after storage at 30°C. for at least one, two or three months. Visible particles are suitablydetected using the 2.9.20. European Pharmacepoeia Monograph (ParticulateContamination: Visible Particles)

Suitably the formulations of the invention are sufficiently stable thatthe concentration of related species remains low upon extended storage.The term “related species” as used herein, refers to any component ofthe protein content formed by a chemical modification of the parentinsulin or insulin analogue, particularly desamido or cyclic imide formsof insulin. Related species are suitably detected by RP-HPLC.

In a preferred embodiment, the formulation of the invention retains atleast 95%, e.g. at least 96%, e.g. at least 97%, e.g. at least 98%native insulin or native insulin analogue (by weight of total protein)after storage at 30° C. for at least one, two or three months. Thepercentage of insulin or insulin analogue (by weight of total protein)may be determined by size-exclusion chromatography or RP-HPLC.

In a preferred embodiment, the formulation of the invention comprises nomore than 4% (by weight of total protein), preferably no more than 2%high molecular weight species after storage at 30° C. for at least one,two or three months.

In a preferred embodiment, the formulation of the invention comprises nomore than 4% (by weight of total protein), preferably no more than 2%A-21 desamido form of the insulin or the insulin analogue after storageat 30° C. for at least one, two or three months.

In preferred embodiments, a composition of the present invention shouldexhibit an increase in high molecular weight species during storagewhich is at least 10% lower, preferably at least 25% lower, morepreferably at least 50% lower, than a composition lacking thepolysorbate 80 but otherwise identical, following storage under the sameconditions and length of time.

In preferred embodiments, a composition of the present invention shouldexhibit an increase in related species during storage which is at least10% lower, preferably at least 25% lower, more preferably at least 50%lower, than a composition lacking the polysorbate 80 but otherwiseidentical, following storage under the same conditions and length oftime.

The speed of action of a formulation of the invention may be determinedin the

Diabetic Pig Pharmacokinetic/Pharmacodynamic Model (see Examples,General Methods). In preferred embodiments, a composition of the presentinvention should exhibit a Tmax (i.e. time to peak insulinconcentration) that is least 10% shorter, preferably at least 20%shorter, more preferably at least 30% shorter than a composition lackingthe chelating agent but otherwise identical, using the model. Inpreferred embodiments, a composition of the present invention shouldexhibit an area under the curve on the pharmacodynamics profile withinthe first 45 minutes after injection that is least 10% greater,preferably at least 20% greater, more preferably at least 30% greaterthan a composition lacking the chelating agent but otherwise identical,using the model.

According to further aspects of the invention, there is provided aformulation of the invention for use in the treatment of a subjectsuffering from diabetes mellitus. There is also provided a method oftreatment of diabetes mellitus which comprises administering to asubject in need thereof an effective amount of a formulation of theinvention.

A typical dose of the composition of the invention is 2-30 U, e.g. 5-15U.

Administration should suitably occur in the window between 15 minutesbefore eating (i.e. before start of a meal) and 15 minutes after eating(i.e. after end of a meal).

An aspect of the invention is a container e.g. made of plastics or glasscontaining one dose or a plurality of doses of the formulation of theinvention. The container can, for example, be a cartridge designed to bea replaceable item for use with an injection device.

The formulations of the invention may suitably be packaged forinjection, especially sub-cutaneous or intramuscular injection.Sub-cutaneous injection is preferred. Injection may be by conventionalsyringe or more preferably via a pen device adapted for use by diabeticsubjects. Exemplary pen devices include the Kwikpen® device and theFlexpen® device.

An aspect of the invention is an injection device, particularly a deviceadapted for subcutaneous or intramuscular injection, for single ormultiple use comprising a container containing one dose or a pluralityof doses of the formulation of the invention together with an injectionneedle. In an embodiment the container is a replaceable cartridge whichcontains a plurality of doses. In an embodiment, the needle isreplaceable e.g. after each occasion of use.

Another aspect of the invention is a medical device comprising areservoir comprising plurality of doses of the formulation of theinvention and a pump adapted for automatic or remote operation such thatupon automatic or remote operation one or more doses of the formulationof the invention is administered to the body e.g. subcutaneously orintramuscularly. Such devices may be worn on the outside of the body orimplanted in the body.

Formulations of the invention may be prepared by mixing the ingredients.For example, the insulin or insulin analogue may be dissolved in anaqueous formulation comprising the other components. Alternatively, theinsulin or insulin analogue may be dissolved in a strong acid (typicallyHCl), after dissolution diluted with an aqueous formulation comprisingthe other components, and then pH adjusted to the desired pH withaddition of alkali (e.g. NaOH). As a variation on this method, a step ofneutralising the acid solution may be performed before the dilution stepand it may then not be necessary to adjust the pH after the dilutionstep (or a small adjustment only may be necessary).

According to another aspect of the invention there is provided a drysolid pharmaceutical composition suitable for reconstitution with anaqueous medium which comprises insulin or an insulin analogue, ioniczinc, a chelating agent and polysorbate 80. Thus, a formulation of theinvention may be prepared by dissolving such a dry solid pharmaceuticalcomposition in an aqueous medium e.g. water or saline. Such a dry solidpharmaceutical composition may be prepared by dehydrating (e.g. freezedrying) a formulation of the invention. The invention also provides acontainer containing one dose or a plurality of doses of such a drysolid pharmaceutical composition.

The invention includes an aqueous liquid pharmaceutical formulationcomprising insulin or an insulin analogue, ionic zinc, a chelating agentand polysorbate 80. In some embodiments, the formulation comprisesinsulin lispro as an insulin analogue. In some embodiments, theformulation comprises insulin aspart as an insulin analogue. In someembodiments, the formulation comprises insulin glulisine as an insulinanalogue. In some embodiments, the formulation comprises recombinanthuman insulin as an insulin. In additional embodiments, the insulin orinsulin analogue is present in the formulation at a concentration of10-1000 U/ml. In additional embodiments, the ionic zinc in theformulation is present at a concentration of more than 0.05% e.g. morethan 0.25% by weight of zinc based on the weight of insulin or insulinanalogue in the formulation. In further embodiments, the ionic zinc ispresent at a concentration of 0.25-1% by weight of zinc based on theweight of insulin or insulin analogue in the formulation. In additionalembodiments, the chelating agent in the formulation has a metal bindingstability constant logK with respect to zinc binding of at least 4.5 asdetermined at 25° C. In some embodiments, the chelating agent is EDTA.In some embodiments, the chelating agent is selected from citrate, EGTA,pyrophosphate, alginate, ethylenediamine and histidine. In otherembodiments, the chelating agent is citrate. In further embodiments, thesource of the citrate of the chelating agent in the formulation iscitric acid. In other embodiments, the chelating agent is histidine. Insome embodiments, the chelating agent is present in the formulation at aconcentration of 0.1-50 mM. In some embodiments, the chelating agent inthe formulation is EDTA at a concentration of 0.1-2 mM. In someembodiments, the chelating agent in the formulation is citrate at aconcentration of 2.5-50 mM. In additional embodiments, the molar ratioof ionic zinc to EDTA as chelating agent in the formulation is in therange 1:0.8 to 1.0:2.0. In additional embodiments, the molar ratio ofionic zinc to citrate as chelating agent is in the range 1:20-1:100. Inadditional embodiments, the polysorbate 80 in the formulation of theinvention is present at a concentration of 1-500 μg/ml. In additionalembodiments, the formulation further comprises an uncharged tonicitymodifier. In some embodiments, the uncharged tonicity modifier isselected from the group consisting of trehalose, mannitol, glycerol or1,2-propanediol. In some embodiments, the uncharged tonicity modifier isglycerol. In additional embodiments, the formulation is isotonic. Inadditional embodiments, the pH of the formulation is in the range 5.5 to9.0. In some embodiments, the pH is in the range 7.0 to 7.5. In furtherembodiments, the pH is in the range 7.6 to 8.0. In additionalembodiments, the formulation, further comprises a preservative. In someembodiments, the preservative is selected from the group consisting ofphenol, m-cresol, chlorocresol, benzyl alcohol, propylparaben,methylparaben, benzalkonium chloride and benzethonium chloride. Inadditional embodiments, the ionic strength of the formulation is lessthan 40 mM. In additional embodiments, the formulation is for use in thetreatment of a subject suffering from diabetes mellitus. In someembodiments, the disclosure provides a method of treatment of diabetesmellitus which comprises administering to a subject in need thereof aneffective amount of a formulation of the invention. In some embodiments,the disclosure provides a container containing one dose or a pluralityof doses of the formulation. In an additional embodiment, the inventionprovides an injection device for single or multiple use comprising acontainer containing one dose or a plurality of doses of the formulationof the invention together with an injection needle. In some embodiments,the invention provides a medical device comprising a reservoircomprising plurality of doses of the formulation and a pump adapted forautomatic or remote operation such that upon automatic or remoteoperation one or more doses of the formulation is administered to thebody. In an additional embodiment, the invention provides a dry solidpharmaceutical composition suitable for reconstitution with an aqueousmedium which comprises insulin or an insulin analogue, ionic zinc, achelating agent and polysorbate 80. In one embodiment, the inventionprovides a method of preparing a formulation of the invention whichcomprises dissolving a dry solid pharmaceutical composition of theinvention in an aqueous medium. In another embodiment, the inventionprovides a method of improving the storage stability of an aqueousliquid pharmaceutical formulation comprising insulin or an insulinanalogue, ionic zinc and a chelating agent which comprises addingpolysorbate 80 to the formulation. In an additional embodiment, theinvention provides the use of polysorbate 80 to improve the storagestability of an aqueous liquid pharmaceutical formulation comprisinginsulin or an insulin analogue, ionic zinc and a chelating agent.

Further aspects of the invention include:

-   [A] An aqueous liquid pharmaceutical formulation comprising (i) an    insulin or an insulin analogue, (ii) ionic zinc, (iii) a chelating    agent at a concentration of 0.1-100 mM e.g. 0.1-50 mM, and (iv)    polysorbate 80 at a concentration of 1-800 μg/ml, e.g. 1-500 μg/ml,    e.g. 5-200 μg/ml, e.g. 5-50 μg/ml, e.g. 20 μg/ml. In some    embodiments, the chelating agent is selected from citrate, EGTA,    pyrophosphate, alginate, ethylenediamine and histidine. In some    embodiments, the chelating agent is citrate e.g. at a concentration    of 2.5-50 mM, e.g. 5-50 mM, e.g. 5-30 mM e.g. 22 mM or 10-50 mM e.g.    30-50 mM e.g. 44 mM. In some embodiments, the chelating agent is    histidine e.g. at a concentration of 2.5-50 mM, e.g. 5-50 mM, e.g.    5-30 mM, e.g. 22 mM, or 10-50 mM, e.g. 30-50 mM, e.g. 44 mM. In some    embodiments, the chelating agent is EDTA e.g. at a concentration of    0.1-2 mM, e.g. 0.2-1 mM, e.g. 0.3-1 mM, e.g. 0.3-0.5 mM, e.g. 0.4    mM. In some embodiments, the ionic zinc is present at a    concentration of more than 0.05% e.g. more than 0.1% e.g. more than    0.2% e.g. 0.25-1% e.g. 0.35-0.75% e.g. 0.45-0.6% by weight of zinc    based on the weight of insulin or insulin analogue in the    formulation. In some embodiments, the ionic zinc is present at a    concentration of more than 0.015 mM, e.g. more than 0.15 mM, e.g.    0.15-0.60 mM, e.g. 0.20-0.45 mM, e.g. 0.25-0.35 mM. In some    embodiments, the ionic zinc is present at a concentration of more    than 1.5 mM, e.g. 1.5-6.0 mM, e.g. 2.0-4.5, e.g. 2.5-3.5 mM. In some    embodiments, the insulin or insulin analogue is present at a    concentration of 10-1000 U/ml, e.g., 50-500 U/ml, 50-200 U/ml e.g.    100 U/ml. In some embodiments, the insulin or insulin analogue is    present at a concentration of 500-1000 U/ml, e.g. 800-1000 U/ml,    e.g. 1000 U/ml. In a first further embodiment, the formulation    comprises the insulin analogue insulin lispro. In a second further    embodiment, the formulation comprises the insulin analogue insulin    aspart. In a third further embodiment, the formulation comprises the    insulin analogue insulin glulisine. In a fourth further embodiment,    the formulation comprises recombinant human insulin.    -   [B] The aqueous liquid pharmaceutical formulation of [A],        further comprising one or more buffers e.g. phosphate such as        sodium phosphate.    -   [C] The aqueous liquid pharmaceutical formulation of [A] or [B],        further comprising one or more preservatives e.g. phenol,        m-cresol, chlorocresol, benzyl alcohol, propylparaben,        methylparaben, benzalkonium chloride or benzethonium chloride.    -   [D] The aqueous liquid pharmaceutical formulation of [A], [B],        or [C], further comprising one or more tonicity modifiers e.g.        glycerol or NaCl.    -   [E] The aqueous liquid pharmaceutical formulation of [A], [B],        [C] or [D], further comprising nicotinamide, nicotinic acid or a        salt thereof In one embodiment, the nicotinamide, nicotinic acid        or salt thereof, is at a concentration in the range of 10-150        mM, preferably in the range 20-100 mM, such as around 80 mM.    -   [F] The aqueous liquid pharmaceutical formulation of [A], [B],        [C], [D] or [E], wherein the ionic strength of the formulation        is less than 40 mM.    -   [G] A method of improving the storage stability of an aqueous        liquid pharmaceutical formulation comprising insulin or an        insulin analogue, ionic zinc and a chelating agent which        comprises adding polysorbate 80 to the formulation.    -   [H] Use of polysorbate 80 to improve the storage stability of an        aqueous liquid pharmaceutical formulation comprising insulin or        an insulin analogue, ionic zinc and a chelating agent.

Aspects of the invention, without limitation, are defined by thefollowing embodiments:

-   -   [1] An aqueous liquid pharmaceutical formulation comprising        insulin or an insulin analogue, ionic zinc, a chelating agent        and polysorbate 80.    -   [2] The formulation according to embodiment [1] comprising        insulin lispro as an insulin analogue.    -   [3] The formulation according to embodiment [1] comprising        insulin aspart as an insulin analogue.    -   [4] The formulation according to embodiment [1] comprising        insulin glulisine as an insulin analogue.    -   [5] The formulation according to embodiment [1] comprising        recombinant human insulin as an insulin.    -   [6] The formulation according to any one of embodiments [1] to        [5], wherein the insulin or insulin analogue is present at a        concentration of 10-1000 U/ml.    -   [7] The formulation according to any one of embodiments [1] to        [6], wherein the ionic zinc is present at a concentration of        more than 0.25% by weight of zinc based on the weight of insulin        or insulin analogue in the formulation.    -   [8] The formulation according to embodiment [7], wherein the        ionic zinc is present at a concentration of 0.25-1% by weight of        zinc based on the weight of insulin or insulin analogue in the        formulation.    -   [9] The formulation according to any one of embodiments [1] to        [8], wherein the chelating agent has a metal binding stability        constant logK with respect to zinc binding of at least 4.5 at        25° C.    -   [10] The formulation according to any one of embodiments [1] to        [9], wherein the chelating agent is EDTA.    -   [11] The formulation according to any one of embodiments [1] to        [9], wherein the chelating agent is selected from citrate, EGTA,        pyrophosphate, alginate, ethylenediamine and histidine.    -   [12] The formulation according to embodiment [11], wherein the        chelating agent is citrate.    -   [13] The formulation according to embodiment [12] wherein the        source of the citrate is citric acid.    -   [14] The formulation according to any of embodiments [1] to        [13], wherein the chelating agent is present at a concentration        of 0.1-50 mM.    -   [15] The formulation according to embodiment [10] wherein EDTA        as chelating agent is present at a concentration of 0.1-2 mM.    -   [16] The formulation according to embodiment [12] or [13]        wherein citrate as chelating agent are present at a        concentration of 2.5-50 mM.    -   [17] The formulation according to any one of embodiments [10],        [14] or [15] wherein the molar ratio of ionic zinc to EDTA as        chelating agent is in the range 1:0.8 to 1.0:2.0.    -   [18] The formulation according to any one of embodiments [11] to        [14] or [16], wherein the molar ratio of ionic zinc to citrate        as chelating agent is in the range 1:20-1:100.    -   [19] The formulation according to any one of embodiments [1] to        [18], wherein the polysorbate 80 is present at a concentration        of 1-500 μg/ml.    -   [20] The formulation according to any one of embodiments [1] to        [19], further comprising an uncharged tonicity modifier.    -   [21] The formulation according to embodiment [20], wherein the        uncharged tonicity modifier is selected from the group        consisting of trehalose, mannitol, glycerol or 1,2-propanediol.    -   [22] The formulation according to embodiment [21], wherein the        uncharged tonicity modifier is glycerol.    -   [23] The formulation according to any one of embodiments [1] to        [22], wherein the composition is isotonic.    -   [24] The formulation according to any one of embodiments [1] to        [23], wherein the pH is in the range [5]5 to [90].    -   [25] The formulation according to embodiment [24], wherein the        pH is in the range 7.0 to 7.5.    -   [26] The formulation according to embodiment [24], wherein the        pH is in the range 7.6 to 8.0.    -   [27] The formulation according to any of embodiments [1] to        [26], further comprising a preservative.    -   [28] The formulation according to embodiment [27], wherein the        preservative is selected from the group consisting of phenol,        m-cresol, chlorocresol, benzyl alcohol, propylparaben,        methylparaben, benzalkonium chloride and benzethonium chloride.    -   [29] The formulation according to any one of embodiments [1] to        [28] wherein the ionic strength of the formulation is less than        40 mM.    -   [30] A formulation according to any one of embodiments [1] to        [29] for use in the treatment of a subject suffering from        diabetes mellitus.    -   [31] A method of treatment of diabetes mellitus which comprises        administering to a subject in need thereof an effective amount        of a formulation according to any one of embodiments [1] to        [29].    -   [32] A container containing one dose or a plurality of doses of        the formulation according to any one of embodiments [1] to [29].    -   [33] An injection device for single or multiple use comprising a        container containing one dose or a plurality of doses of the        formulation according to any one of embodiments [1] to [29]        together with an injection needle.    -   [34] A medical device comprising a reservoir comprising        plurality of doses of the formulation according to any one of        embodiments [1] to [29] and a pump adapted for automatic or        remote operation such that upon automatic or remote operation        one or more doses of the formulation is administered to the        body.    -   [35] A dry solid pharmaceutical composition suitable for        reconstitution with an aqueous medium which comprises insulin or        an insulin analogue, ionic zinc, a chelating agent and        polysorbate 80.    -   [36] A method of preparing a formulation according to any one of        embodiments [1] to [29] which comprises dissolving a dry solid        pharmaceutical composition according to embodiment [35] in an        aqueous medium.    -   [37] A method of improving the storage stability of an aqueous        liquid pharmaceutical formulation comprising insulin or an        insulin analogue, ionic zinc and a chelating agent which        comprises adding polysorbate 80 to the formulation.    -   [38] Use of polysorbate 80 to improve the storage stability of        an aqueous liquid pharmaceutical formulation comprising insulin        or an insulin analogue, ionic zinc and a chelating agent.

Formulations of the invention are expected to have one or more of thefollowing advantageous properties:

-   -   rapid speed of action, typically faster than normal human        insulin, upon administration to a subject;    -   good physical stability upon storage, especially as measured by        the amount of HMWS or visual detection of particles;    -   good chemical stability upon storage, especially as measured by        the amount of related products e.g. products of deamidation.

EXAMPLES General Methods Size Exclusion Chromatography

Ultra-high performance size exclusion chromatography of insulinpreparations was performed using the Waters ACQUITY H-class Bio UPLC®system with a 1.7 μm Ethylene Bridged Hybrid 125 Å pore packing materialin a 300 mm by 4.6 mm column. The column was equilibrated in 0.65 mg/mlL-arginine, 20% v/v acetonitrile, 15%v/v glacial acetic acid mobilephase and 10 μl of sample, acidified with 0.01M HCl, was analysed at 0.4mL/min, with 276 nm UV detection. All analyses were performed at ambienttemperature.

Reversed-Phase

Ultra-high performance reverse phase chromatography was performed usingthe Waters ACQUITY H-class Bio UPLC® system with a 1.7 μm EthyleneBridged Hybrid particle, 130 A pore resin trifunctionally immobilisedwith a Cl₈ ligand in a 50 mm by 2.1 mm column. Insulin samples werebound in an 82%w/v Na2SO₄, 18% v/v acetonitrile, pH 2.3 mobile phase andeluted in 50% w/v Na2SO_(4, 50)% v/v acetonitrile gradient flow. 2μl ofsample was acidified with 0.01M HCland analysed at 0.61 mL/min, with 214nm UV detection. All analyses were performed at 40° C.

The Diabetic Pig Pharmacokinetic/Pharmacodynamic Model: Method fordetermining speed of action:

10-15 male diabetic Yucatan miniature pigs are used. Pigs are injectedsubcutaneously with a sample of the test formulation and blood is taken(1 or 2 ml) at the following time-points (min) with respect to theinjection: −30 (or -15), 0, 5, 10, 15, 30, 45, 60, 75, 90, 105, 120,150, 180, 240, 360. For pharmacodynamics profile, serum is analysed forglucose (using a commercially available glucometer). For pharmacokineticprofile, insulin concentration is determined in the serum using animmunoassay.

Visual assessment

Visible particles are suitably detected using the 2.9.20. European

Pharmacepoeia Monograph (Particulate Contamination: Visible Particles).The apparatus required consists of a viewing station comprising:

-   -   a matt black panel of appropriate size held in a vertical        position    -   a non-glare white panel of appropriate size held in a vertical        position next to the black panel    -   an adjustable lampholder fitted with a suitable, shaded,        white-light source and with a suitable light diffuser (a viewing        illuminator containing two 13 W fluorescent tubes, each 525 mm        in length, is suitable). The intensity of illumination at the        viewing point is maintained between 2000 lux and 3750 lux.

Any adherent labels are removed from the container and the outsidewashed and dried. The container is gently swirled or inverted, ensuringthat air bubbles are not introduced, and observed for about 5 s in frontof the white panel. The procedure is repeated in front of the blackpanel. The presence of any particles is recorded.

The visual scores are ranked as follows:

Visual Assessment Scoring Method A

Visual score 1: clear solution free of particles

Visual score 2: slight particle formation

Visual score 3: more significant precipitation

Visual Assessment Scoring Method B

Visual score 1: Clear solution, virtually free of particles

Visual score 2: ˜5 very small particles

Visual score 3: ˜10-20 very small particles

Visual score 4: 20-50 particles, including larger particles

Visual score 5: >50 particles, including larger particles

Whilst the particles in samples with visual scores 4 and 5 are clearlydetectable on casual visual assessment under normal light, samples withvisual score 1-3 generally appear as clear solutions on the sameassessment. Samples with visual scores 1-3 are considered to be “Pass”;samples with visual score 4-5 are considered to be “Fail”.

Example 1 Example Formulations

The following example formulations may be prepared:

Example 1a:

Insulin lispro 100 U/ml EDTA 0.4 mM Sodium phosphate 2 mM Glycerol 173mM m-Cresol 29 mM Ionic zinc (as ZnCl₂) 0.3 mM Polysorbate 80 20 μg/mlWater for injection qs Residual NaCl Acidification and subsequentneutralisation during preparation results in formation of 2-4 mM NaCl pHadjusted to 7.4

Example 1b:

Insulin lispro 500 U/ml EDTA 0.4 mM Sodium phosphate 2 mM Glycerol 173mM m-Cresol 29 mM Ionic zinc (as ZnCl₂) 0.3 mM Polysorbate 80 20 μg/mlWater for injection qs Residual NaCl Acidification and subsequentneutralisation during preparation results in formation of 2-4 mM NaCl pHadjusted to 7.4

Example 1c:

Insulin lispro 100 U/ml EDTA 0.6 mM Sodium phosphate 2 mM Glycerol 173mM m-Cresol 29 mM Ionic zinc (as ZnCl₂) 0.3 mM Polysorbate 80 20 μg/mlWater for injection qs Residual NaCl Acidification and subsequentneutralisation during preparation results in formation of 2-4 mM NaCl pHadjusted to 7.4Method for preparation for the above formulations:

Insulin powder is added to water and HCl is added until the powder isfully dissolved (pH has to be <3 in order to achieve full dissolution).ZnCl₂ is added to the required level. Once dissolved, pH is adjusted toapproximately 7 and volume is adjusted with water so that the insulinconcentration is 2× the required concentration. The composition is thenmixed 1:1 (v/v) with a mixture of additional excipients (all at 2× therequired concentration).

Example 2 Stability of Formulations of the Invention

The effect of different surfactants was tested on stability of insulinlispro (100 U/ml) in the presence of 0.4 mM EDTA. The visual assessmentmethod described in General Methods was used. All compositionscontained: glycerol (173 mM), m-cresol (29 mM), sodium phosphate (2 mM)and zinc chloride (0.3 mM) and were adjusted to pH 7.4. The controlformulation did not contain EDTA; all other formulations contained 0.4mM EDTA.

The results below (Table 1) show that presence of EDTA in the controlformulation resulted in precipitation following incubation at 30° C. for4 weeks. In addition, the size of the main peak (corresponding to intactinsulin) was reduced both on the size-exclusion chromatogram and onRP-HPLC chromatogram compared with the EDTA-free sample. The presence ofpolysorbate 80 prevented these adverse changes, resulting in stabilitythat was almost comparable with the EDTA-free control. Most of the othersamples that contained alternative surfactants also precipitated, someof those to a degree that prevented SEC and RP-HPLC measurement. Somesurfactants (dodecyl maltoside, benzethonium chloride and benzalkoniumchloride) prevented precipitation, but did not prevent the reduction insize of the main peak on SEC and RP-HPLC chromatograms.

TABLE 1 Visual assessment SEC main peak RP-HPLC main peak Surfactant (4weeks at 30° C.) (4 weeks at 30° C.) (4 weeks at 30° C.) Control (noEDTA, Clear 98.54% 97.70% no surfactant) No surfactant (i.e.Precipitated 96.72% 95.78% control + 0.4 mM EDTA) Polysorbate 80 Clear98.10% 97.24% (20 μg/ml) Polysorbate 20 Precipitated Not analysed dueNot analysed due to (20 μg/ml) to excessive excessive precipitationprecipitation Polysorbate 40 Precipitated Not analysed due Not analyseddue to (20 μg/ml) to excessive excessive precipitation precipitationPolysorbate 60 Precipitated Not analysed due Not analysed due to (20μg/ml) to excessive excessive precipitation precipitation DodecylPrecipitated Not analysed due Not analysed due to maltoside to excessiveexcessive (0.1 mg/ml) precipitation precipitation Dodecyl Particlesobserved 96.08% 95.71% maltoside (0.5 mg/ml) Decyl Precipitated Notanalysed due Not analysed due to glucopyranoside to excessive excessive(0.1 mg/ml) precipitation precipitation Octyl Precipitated Not analyseddue Not analysed due to thiogluco- to excessive excessive pyranosideprecipitation precipitation Lauryldi- Particles observed Not analyseddue Not analysed due to methylamine to excessive excessive oxideprecipitation precipitation (0.1 mg/ml) Benzethonium Clear 96.71% 95.78%chloride (20 μg/ml) Benzalkonium Clear 94.47% 95.36% chloride (20 μg/ml)

The above data demonstrates the uniqueness of polysorbate 80 amongstother surfactants,including other polysorbates. Of the formulationstested, only the formulations of the invention showed acceptablestability after storage at 30° C. for 4 weeks.

Example 3 Stability of Insulin Aspart Formulations in the Presence ofCitrate and Polysorbate 80

The effect of polysorbate 80 on stability of insulin aspart (100 U/ml)in the presence of 22 mM sodium citrate was tested as described inGeneral Methods using Visual Assessment Scoring Method A. Allcompositions contained: sodium chloride (150 mM), phenol (15.9 mM),m-cresol (15.9 mM), sodium phosphate (2 mM) ionic zinc (19.7 μg/ml,excluding counter-anion, as ZnCl₂) and were adjusted to pH 7.4. Thecontrol formulation did not contain sodium citrate; all otherformulations contained 22 mM sodium citrate. The results (Table 2) belowshow that presence of sodium citrate to the control formulation resultedin rapid particle formation at 30° C. The presence of polysorbate 80 ledto a considerable reduction in the rate of particle formation.

TABLE 2 Visual scores of insulin aspart compositions following storageat 30° C. using Visual Assessment Scoring Method A. Extent of visibleprecipitation is graded on a scale 1-3; 1 = clear solution free ofvisible particles; 2 = slight particle formation, 3 = more significantprecipitation. 0 days 4 days 7 days 14 days 28 days Control (i.e. nocitrate, 1 1 1 1 1 no surfactant) 22 mM citrate 1 2 3 3 3 22 mMcitrate + 50 1 1 2 2 2 μg/ml polysorbate 80

Example 4 Effect of Polysorbate 80 on the Stability of Insulin Aspart inthe Presence of Trisodium Citrate, L-histidine and Pyrophosphate

Stability of insulin aspart (100 U/ml) was investigated in compositionscomprising trisodium citrate (22 mM), L-histidine (10 mM) orpyrophosphate (5 mM), both in the presence and in the absence ofpolysorbate 80. Polysorbate 20 and poloxamer 188 were also tested ascomparators. All compositions tested further comprised sodium chloride(150 mM), phenol (15.9 mM), m-cresol (15.9 mM), sodium phosphate (2 mM),ionic zinc (19.7 μg/ml, excluding counter-anion, as ZnCl₂) and wereadjusted to pH 7.4.

It was shown (Table 3) that the presence of trisodium citrate,L-histidine or pyrophosphate increased considerably the rate of particleformation in formulations of insulin aspart, using the Visual AssessmentScoring Method B. The presence of polysorbate 80 showed a stabilisingeffect. The ability of poloxamer 188 to mitigate the increase inparticle formation rate was shown to be worse than that of polysorbate80. Polysorbate 20 was not effective at all in this experiment.

TABLE 3 Visual scores of insulin aspart (100 U/ml) formulations usingVisual Assessment Scoring Method B following storage at 30° C.Surfactant (all at Chelating agent 50 μg/ml) 0 days 4 days 7 days 14days 28 days None None 1 1 1 1 1 Trisodium citrate (22 mM) None 1 3 4 55 Trisodium citrate (22 mM) Polysorbate 80 1 1 3 3 3 Trisodium citrate(22 mM) Polysorbate 20 1 3 4 5 5 Trisodium citrate (22 mM) Poloxamer 1881 2 4 5 5 L-Histidine (10 mM) None 1 4 5 5 5 L-Histidine (10 mM)Polysorbate 80 1 4 4 4 5 L-Histidine (10 mM) Polysorbate 20 1 4 5 5 5L-Histidine (10 mM) Poloxamer 188 1 4 4 5 5 Pyrophosphate (5 mM) None 15 5 5 5 Pyrophosphate (5 mM) Polysorbate 80 1 4 5 5 5 Pyrophosphate (5mM) Polysorbate 20 1 5 5 5 5 Pyrophosphate (5 mM) Poloxamer 188 1 5 5 55

Example 5 Effect of Polysorbate 80 on the Stability of Insulin Lispro inthe Presence of Citric Acid

Stability of insulin lispro (100 U/ml) was investigated in formulationscomprising citric acid (22 mM), both in the presence and in the absenceof polysorbate 80. Polysorbate 20 and poloxamer 188 were also tested ascomparators. All formulations contained: phenol (15.9 mM), m-cresol(15.9 mM), sodium phosphate (2 mM), ionic zinc (19.7 μg/ml, excludingcounter-anion, as ZnCl₂) and were adjusted to pH 7.8. Formulationscontained glycerol (174 mM) as a tonicity modifier.

It was shown (Table 4) that the presence of citric acid (22 mM) resultedin an increased formation of particles in compositions of insulin lisproin the absence of polysorbate 80 or poloxamer 188, using the VisualAssessment Scoring Method B. The presence of polysorbate 80 appeared tomitigate the destabilising effect. The stabilising effects ofpolysorbate 20 and poloxamer 188 were notably weaker than that ofpolysorbate 80

TABLE 4 Visual scores of insulin lispro (100 U/ml) formulations usingVisual Assessment Scoring Method B following storage at indicatedtemperatures. Citric acid Tonicity T = 0 2-8° C. 30° C. 30° C. 37° C.(mM) Surfactant modifier weeks (8 weeks) (4 weeks) (8 weeks) (4 weeks) 0mM None Glycerol 1 1 1 1 2 (174 mM) 22 mM None Glycerol 1 1 4 5 5 (174mM) 22 mM Polysorbate 80 Glycerol 1 1 2 2 3 (50 μg/ml) (174 mM) 22 mMPolysorbate 20 Glycerol 1 1 3 4 4 (50 μg/ml) (174 mM) 22 mM Poloxamer188 Glycerol 1 1 4 4 5 (50 μg/ml) (174 mM)

Example 6 Effect of Polysorbate 80 on the Stability of Insulin Aspart(1000 U/ml) in the Presence of Trisodium Citrate, L-histidine andPyrophosphate

Stability of insulin aspart (1000 U/ml) was investigated in formulationscomprising trisodium citrate (44 mM), L-histidine (22 mM) orpyrophosphate (22 mM), both in the presence and in the absence ofpolysorbate 80. All compositions further comprised phenol (15.9 mM),m-cresol (15.9 mM), sodium phosphate (2 mM), glycerol (174 mM), sodiumchloride (10 mM) and ionic zinc (197 μp/ml, excluding counter-anion, asZnCl₂) and were adjusted to pH 7.4.

It was shown (Table 5) that the presence of trisodium citrate,L-histidine or pyrophosphate resulted in a considerable increase in therate of particle formation of insulin aspart, using the VisualAssessment Scoring Method B. The presence of polysorbate 80 showed astabilising effect.

TABLE 5 Visual scores of insulin aspart (1000 U/ml) formulations usingVisual Assessment Scoring Method B following storage at indicatedtemperatures. Ionic Chelating strength* T = 0 2-8° C. 30° C. 30° C. 37°C. agent Surfactant (mM) weeks (12 weeks) (4 weeks) (12 weeks) (4 weeks)None None 24.16 1 1 2 2 3 Citrate None 24.16 1 2 4 5 5 (44 mM) CitratePolysorbate 80 24.16 1 2 1 3 5 (44 mM) (50 μg/ml) Histidine None 24.16 12 4 5 5 (22 mM) Histidine Polysorbate 24.16 1 2 4 5 4 (22 mM) 80 (50μg/ml) Pyrophosphate None 24.16 1 3 5 5 5 (22 mM) PyrophosphatePolysorbate 24.16 1 1 4 5 5 (22 mM) 80 (50 μg/ml) *ionic strengthcalculation takes into account all ions in the formulation except forthe zinc binding species (trisodium citrate, L-histidine orpyrophosphate) and the insulin compound using formula I.

Example 7 Investigation of the Optimal Concentration of Polysorbate 80on the Stability of Insulin Aspart (1000 U/ml) in the Presence ofdifferent Concentrations of Citric Acid

The stability of insulin aspart (1000 U/ml) was investigated in thepresence of different concentrations of citric acid and differentconcentrations of polysorbate 80. All formulations tested furthercomprised phenol (15.9 mM), m-cresol (15.9 mM), sodium phosphate (2 mM),glycerol (174 mM) and ionic zinc (197 μp/ml, excluding counter-anion, asZnCl₂) and were adjusted to pH 7.8. Three concentrations of citric acid(44, 66 and 88 mM) and four concentrations of each non-ionic surfactantwere tested as well as corresponding surfactant-free compositions.

The rate of particle formation in formulations of insulin aspart (1000U/ml) was found to be proportional to citric acid concentration in therange between 44 and 88 mM and, thus, the lower concentration of citricacid (44 mM is more suitable in this experiment (Table 6). The presenceof polysorbate 80 led to a reduction in the rate of particle formation.The higher concentrations (0.3 and 0.5 mg/ml) of polysorbate 80 showed agreater ability to reduce the particle formation rate than the lowerconcentrations (0.05 and 0.1 mg/ml).

TABLE 6 Visual scores of insulin aspart (1000 U/ml) formulations usingVisual Assessment Scoring Method B following storage at indicatedtemperatures. Ionic Citric Polysorbate strength* T = 0 2-8° C. 30° C.30° C. 37° C. acid 80 (mg/ml) (mM) weeks (8 weeks) (4 weeks) (8 weeks)(4 weeks) 44 mM 0 14.84 1 3 4 5 5 44 mM 0.05 14.84 1 3 2 3 4 44 mM 0.114.84 1 2 2 3 4 44 mM 0.3 14.84 1 2 2 3 4 44 mM 0.5 14.84 1 1 1 3 4 66mM 0 14.84 1 5 5 5 5 66 mM 0.05 14.84 1 5 4 5 5 66 mM 0.1 14.84 1 5 4 55 66 mM 0.3 14.84 1 4 3 4 4 66 mM 0.5 14.84 1 4 4 5 5 88 mM 0 14.84 1 55 5 5 88 mM 0.05 14.84 1 5 4 5 5 88 mM 0.1 14.84 1 5 4 4 5 88 mM 0.314.84 1 5 3 4 5 88 mM 0.5 14.84 1 5 3 5 5 *ionic strength calculationtakes into account all ions in the formulation except for the zincbinding species (citric acid) and the insulin compound using formula I.

Abbreviations:

-   EDTA ethylenediaminetetraacetate-   EGTA ethyleneglycoltetraacetate-   HPLC high performance liquid chromatography-   HMWS high molecular weight species-   RP reverse phase-   SEC size-exclusion chromatography

Throughout the specification and the claims which follow, unless thecontext requires otherwise, the word ‘comprise’, and variations such as‘comprises’ and ‘comprising’, will be understood to imply the inclusionof a stated integer, step, group of integers or group of steps but notto the exclusion of any other integer, step, group of integers or groupof steps.

The term “and/or” as used in a phrase such as “A and/or B” herein isintended to include both A and B; A or B; A (alone); and B (alone).Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C”is intended to encompass each of the following embodiments: A, B, and C;A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A(alone); B (alone); and C (alone).

All publications, patents, patent applications, internet sites, andaccession numbers/database sequences (including both polynucleotide andpolypeptide sequences) cited are herein incorporated by reference intheir entirety for all purposes to the same extent as if each individualpublication, patent, patent application, internet site, or accessionnumber/database sequence were specifically and individually indicated tobe so incorporated by reference.

Sequence Listing:

SEQ ID NO: 1:  GIVEQCCTSICSLYQLENYCN SEQ ID NO: 2: FVNQHLCGSHLVEALYLVCGERGFFYTPKT SEQ ID NO: 3: FVNQHLCGSHLVEALYLVCGERGFFYTKPT SEQ ID NO: 4: FVNQHLCGSHLVEALYLVCGERGFFYTDKT SEQ ID NO: 5: FVKQHLCGSHLVEALYLVCGERGFFYTPET

1. An aqueous liquid pharmaceutical formulation comprising insulin or aninsulin analogue, ionic zinc, a chelating agent and polysorbate
 80. 2.The formulation according to claim 1 comprising insulin lispro as aninsulin analogue.
 3. The formulation according to claim 1 comprisinginsulin aspart as an insulin analogue.
 4. The formulation according toclaim 1 comprising insulin glulisine as an insulin analogue.
 5. Theformulation according to claim 1 comprising recombinant human insulin asan insulin.
 6. The formulation according to claim 1, wherein the insulinor insulin analogue is present at a concentration of 10-1000 U/ml. 7.The formulation according to claim 1, wherein the ionic zinc is presentat a concentration of more than 0.25% by weight of zinc based on theweight of insulin or insulin analogue in the formulation.
 8. Theformulation according to claim 7, wherein the ionic zinc is present at aconcentration of 0.25-1% by weight of zinc based on the weight ofinsulin or insulin analogue in the formulation.
 9. The formulationaccording to claim 1, wherein the chelating agent has a metal bindingstability constant logK with respect to zinc binding of at least 4.5 at25° C.
 10. The formulation according to claim 1, wherein the chelatingagent is EDTA.
 11. The formulation according to claim 1, wherein thechelating agent is selected from citrate, EGTA, pyrophosphate, alginate,ethylenediamine and histidine.
 12. The formulation according to claim11, wherein the chelating agent is citrate.
 13. The formulationaccording to claim 12 wherein the source of the citrate is citric acid.14. The formulation according to claim 1, wherein the chelating agent ispresent at a concentration of 0.1-50 mM.
 15. The formulation accordingto claim 10 wherein EDTA as chelating agent is present at aconcentration of 0.1-2 mM.
 16. The formulation according to claim 12wherein citrate as chelating agent are present at a concentration of2.5-50 mM.
 17. The formulation according to claim 10, wherein the molarratio of ionic zinc to EDTA as chelating agent is in the range 1:0.8 to1.0:2.0.
 18. The formulation according to claim 11, wherein the molarratio of ionic zinc to citrate as chelating agent is in the range1:20-1:100.
 19. The formulation according to claim 1, wherein thepolysorbate 80 is present at a concentration of 1-500 μg/ml.
 20. Theformulation according to claim 1, further comprising an unchargedtonicity modifier.
 21. The formulation according to claim 20, whereinthe uncharged tonicity modifier is selected from the group consisting oftrehalose, mannitol, glycerol or 1,2-propanediol.
 22. The formulationaccording to claim 21, wherein the uncharged tonicity modifier isglycerol.
 23. The formulation according to claim 1, wherein thecomposition is isotonic.
 24. The formulation according to claim 1,wherein the pH is in the range 5.5 to 9.0.
 25. The formulation accordingto claim 24, wherein the pH is in the range 7.0 to 7.5.
 26. Theformulation according to claim 24, wherein the pH is in the range 7.6 to8.0.
 27. The formulation according to claim 1, further comprising apreservative.
 28. The formulation according to claim 27, wherein thepreservative is selected from the group consisting of phenol, m-cresol,chlorocresol, benzyl alcohol, propylparaben, methylparaben, benzalkoniumchloride and benzethonium chloride.
 29. The formulation according toclaim 1, wherein the ionic strength of the formulation is less than 40mM.
 30. A method of treatment of diabetes mellitus which comprisesadministering to a subject in need thereof an effective amount of aformulation according to claim
 1. 31. An injection device for single ormultiple use comprising a container containing one dose or a pluralityof doses of the formulation according to claim 1 together with aninjection needle.